Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

Mouse Genetics Studies on Molecular Mechanisms Underlying Bone andCartilage Disorders(Submitted for the 2009 Kappa Delta Award)

Hiroshi Kawaguchi, Toru Akune, Takashi Yamada, Naoshi Ogata

Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo,
Tokyo 113-8655, Japan.

Principal Investigator Information:
Hiroshi Kawaguchi, M.D., Ph.D.
Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo,
Hongo 7-3-1, Bunkyo, Tokyo 113-8655, Japan
Phone: (+81)-33815-5411 (ext. 30473); Fax: (+81)-33818-4082;
Date of birth: January 26, 1959
Organization to which the investigator belongs: ORS

1

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

Contents

Manuscript
Abstract

3

Introduction

4

PPARγ as an intracellular molecule regulating age-related osteoporosis

4

IGF-I / IRS signal regulating age-related osteoporosis

6

Insulin signal in OPLL

7

IRS-1 signal in PTH osteoanabolic action and bone frcature healing

9

Akt1 signal in bone formation and resorption

11

Runx2 regulating cartilage degradation in OA

12Carminerin regulating osteophyte formation in OA

15

Conclusion

18

References

19

Supporting Documents
Summary of clinical relevance28

Disclosure statements

29

Summary of investigators’ work and roles in the manuscript

30

CVs for each author

37

2

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

Abstract
This paper summarizes our mouse genetics studies on the molecular backgrounds ofrepresentative degenerative skeletal diosrders: osteoporosis, ossification of the posterior
longitudinal ligament of the spine (OPLL), bone fracture healing, and osteoarthritis (OA).
By analyzing deficient mice, PPARγ, a key adipogenesis molecule intrinsic to bone marrowprogenitors, was shown to be involved in age-related osteoporosis. Studies on deficient mice and
OPLL patients revealed that insulin and insulin-like growth factor-I (IGF-I) are potent bone
anabolic factors through the balance of distinct signals of the two adaptor molecules, insulin
receptor substrate (IRS)-1 and IRS-2: IRS-1 for maintenance of bone turnover by up-regulating
anabolic and catabolic functions of osteoblasts, while IRS-2 for retaining the predominance of
the anabolic function over the catabolic function. IRS-1 was also essentil for the osteoanabolic
action of parathyroid hormone (PTH) and bone fracture healing. The IRS-1 action was mediatedby Akt1, which was shown to be important in both osteoblasts and osteoclasts to maintain bonemass and turnover.
In our original experimental knee OA models in mice, hypertrophic differentiation ofchondrocytes was induced in the joint cartilage during OA progression. Heterozygous deficient
mice of Runx2 exhibited less susceptibility to cartilage degradation. In the meantime, carminerin,a novel chondrocyte-specific protein which we identified as a chondrocyte calcification factor,
was shown to be important for OA osteophyte formation by analyses of the deficient mice.
Our mouse genetics studies revealed the importance of PPARγ, insulin-IGF-I / IRS-1-IRS-2/Akt1 signal, Runx2, and carminerin in bone and cartilage under physiological and pathologicalconditions. These molecules could be therapeutic targets for the skeletal disorders.

3Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

Introduction
Due to the rapidly increasing number of elderly individuals today, degenerative skeletal diosrders
are now considered a major public health issue causing chronic disability in most developed
countries. Among them, osteoporosis and osteoarthritis (OA) are the two representative disorders
worldwide with strong social impacts.1 Ossification of the posterior longitudinal ligament of thespine (OPLL) with a prevalence of 2-4% in Asia and somewhat lower in other countries is adisorder that causes severe tetraparesis.2 Despite significant social demand for more informationof the etiology of the disorders, the molecular mechanisms have not yet been fully elucidated.
The mouse genetics approach is now considered to be a useful and efficient method due to recent
progression of gene manupulating techniques in vivo. This manuscript introduces our recent
mouse genetics studies attempting to elucidate the molecular mechanisms underlying the bone
and cartilage disorders.

PPARγ as an intracellular molecule regulating age-related osteoporosis
Ageing is one of the major causes of osteoporosis, and the underlying mechanisms include
intracellular and extracellular signals of osteoblastic cells. As an intracellular molecule regulating
age-related osteoporosis, Runx2, a key transcription factor for osteoblast differentiation, is themost probable candidate, since the expression is reported to be decreased during cellular ageingof osteoblasts in vitro.3 However, there is no in vivo evidence of its contribution to bone loss
with ageing.
Osteoblasts and adipocytes are known to share a common progenitor: multipotential
mesenchymal cells in bone marrow, being driven by respective key molecules Runx2 and PPARγ

4

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders(Fig. 1A).4 In addition, ageing isAassociated with a reciprocal
decrease of osteogenesis and an

Runx2

Bone marrowmesenchymal
precursors

Adipocytes

PPARγ

B

Femur
8

C

Tibia

52

8

WT

PPARγ+/–

52 w

increase of adipogenesis in bonemarrow.5 Hence, we investigated the

Osteoblasts

OilredWTrole of PPARγ in bone metabolism
by analyzing heterozygous

ALP
+/–

PPARγ-deficient (PPARγ+/–)mice,

since the homozygous

deficient (PPARγ–/–) mice wereembryonically lethal.8 Although

12
BV/TV (%)

6.7

*

*

9

**

exhibited higher bone mass than thewild-type (WT) littermates,

vonKossa

WT
PPARγ+/–

38

16

they showed no abnormality inmajor organs, PPARγ+/– mice

*

6

0

Alizarin
-red

26
Age

52 w

Figure 1. (A) A schema of differentiation of osteoblasts andadipocytes from the same progenitor cells. (B) Radiographs ofwild-type (WT) and PPARγ+/– bones at 8 and 52 weeks of age,and time course of trabecular bone volume (BV/TV) at the distal
femurs measured on CT images. *P<0.05, **P<0.01 vs. WT. (C)Adipogenesis by oil-red staining and osteogenesis by alkalinephosphatase (ALP), Alizarin-red, and von-Kossa stainings incultures of WT and PPARγ+/– bone marrow cells.

especially at older ages (Fig. 1B), indicating the involvement of the PPARγ signal in thepathophysiology of age-related osteoporosis.6,7 Ex vivo cultures of bone marrow cells showedthat PPARγ haploinsufficiency caused not only a decrease in the number of adipocytes, but also
an increase of osteoblasts (Fig. 1C), indicating that PPARγ signal in marrow progenitors
functions as a potent suppressor of commitment to osteoblastic lineage. Clinical involvement of
PPARγ in bone loss was confirmed by an association study between the human gene
polymorphism and bone density in Japanese postmenopausal women.9

5

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

IGF-I / IRS signal regulating age-related osteoporosis
There are several cytokines / growth factors that function as extracellular signals regulating
age-related osteoporosis,10 and among them IGF-I is the most probable candidate since the serum
level is positively correlated with bone density of aged populations.11 Patients with Laron
syndrome caused by IGF-I deficiency also exhibit severe osteoporosis,12 indicating that IGF-I is apotent bone anabolic factor clinically.
A

A

WT

WT

IRS-1–/–
IRS-2–/–

B

BV/TV (%)

8

WT
IRS-1–/–

4
0
9

BV/TV
(%)

8

Ob.S / BS

8

6
4

0
Oc.N / B.Pm

08(cells / 10 cm)

4

ES / BSMAR(/ day)3

BFR / BS
(mm2/cm2/yr)

1

0
WT
IRS-2–/–

4

2

1

0

3

3
2

BFR / BS
(mm2/cm2/yr)

(%)

100

B

0
Oc.N / B.Pm

400 (cells/10 cm)

ES / BS
6 (%)
4

200

(%)

2

50

4
0

0

0

Figure 2. (A) 3D-CT of distal femurs of
WT and IRS-1–/– littermates at 8 weeks
of age. (B) Trabecular bone volume(BV/TV) and bone histomorphometricanalysis of the proximal tibiae. Both boneformation parameters (Ob.S/BS andBFR/BS) and bone resorption parameters(Oc.N/B.Pm and ES/BS) were decreasedin the IRS-1–/–.

0

Figure 3. (A) Radiographs of femurs and tibias, and 3DCT of distal femurs of WT and IRS-2–/– littermates at 8
weeks of age. (B) Trabecular bone volume (BV/TV) andbone histomorphometric analysis of the proximal tibiae.
Bone formation parameters (MAR and BFR/BS) weredecreased while bone resorption parameters (Oc.N/B.Pm
and ES/BS) were increased in the IRS-2–/–.

6

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

IGF-I as well as insulin initiate cellular responses by binding to their respective cell-surface
receptors, and then activate essential adaptor molecule insulin receptor substrate (IRS) followedby downstream signaling pathways like phosphatidylinositol-3 kinase (PI3K)/Akt andmitogen-activated protein kinases (MAPKs).13 Among the IRS family, we found that IRS-1 andIRS-2 are expressed in bone.14,15 Our further studies on mice lacking IRS-1 (IRS–1–/–) or IRS-2(IRS–2–/–) revealed that these mice exhibited severe osteopenia with distinct mechanisms:
IRS-1–/– mice showed a low bone turnover in which both bone formation and resorption weredecreased (Fig. 2),14 whereas IRS-2–/– mice showed an uncoupling status with decreased boneformation and increased bone resorption (Fig. 3).15 It therefore seems that IRS-1 is important for
maintaining bone turnover, while IRS-2 for remodeling balance retaining the predominance ofanabolic function over
IGF-I / insulin

catabolic function of

Receptor
Osteoblasts

osteoblasts. IGF-I and
IRS-1

insulin may up-regulatebone formation without

IRS-2
Anabolic function

BoneturnoverCatabolic function

Remodelingbalanceaffecting bone resorption
Bone Formation

through the balance of theBone Resorption

two IRS signals inosteoblasts (Fig. 4).16~

Figure 4. A schema of mechanism underlying the osteoanabolic functionof IGF-I and insulin via distinct functions of IRS-2 and IRS-2 inosteoblasts.

Insulin signal in OPLL
Patients with type 1 diabetes with decreased insulin level are often associated with7

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disordersA

17

vertebrae

osteoporosis, probably via theOPLLextentsuppression of IRS-1 and IRS-2
signals above. Besides

5
6
HbA1c (%)
r=0.67, p<0.0001
y=1.47+0.230x

OPLL
extent 100

the relationship between
B

0

10
20
Fasting insulin (µU / mL)

30 0

20
40
60
Insulinogenic index (U / mol)vertebrae
Insulinogenic index

controversial, most OPLL patients

type 2 diabetes. Our clinical4

r=0.35, p=0.012
y=2.93+0.251x

20

other skeletal disorders. Although

18

4
5
6
7
8
Fasting glucose (mmol / L)vertebrae

signal may possibly be involved inhave been reported to accompany

r=-0.07, p=0.641

100osteoporosis, the insulin/IGF-I

osteoporosis and type 2 diabetes is

r=-0.20, p=0.151

20>= Average< Average

20

OPLL
extent10

study examining the relationshipbetween the extent of ossificationand glucose intolerance in OPLL
patients revealed that the insulin
secretory response, but not the0

NGT

Ossification

Insulindiabetes, was associated with theLiver, fat,muscleOsteoprogenitors(-)

confirming the clinical

IRS-1–/– & IRS-2–/– mice

Insulinosteoanabolic action of insulin
under the pathological condition.

DM

OPLL patients(-)

severity of glucose intolerance orossification severity (Fig. 5),19,20

IGR

Figure 5. (A) Correlation between OPLL extent and serum levels of
diabetes-related factors in OPLL patients (n=52). (B) OPLL extent ofpatients with three grades of diabetes severity: non-glucose tolerance(NGT), intermediate glucose resistance (IGR), and diabetes mellitus(DM). Patients with insulinogenic index equal to or higher than the
average are shown as red circles and those less than that are shown as
blue circles.

Liver, fat,muscleOsteoporosis
Osteoblasts

Figure 6. A schema of mechanisms underlying the ossification in
OPLL patients, and osteoporosis in IRS-1–/– and IRS-2–/– mice.

8

7

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

Taking the clinical and mouse genetics studies together, in OPLL patients with type 2
diabetes the up-regulation of insulin production as a feedback mechanism of the impairment of
insulin sensitivity in the target cells may stimulate osteoprogenitor cells in the ligament to induce
ossification. Contrarily, in IRS-1–/– and IRS-2–/– mice even the increased insulin may not affectosteoblasts, resulting in osteoporosis (Fig. 6).
A

rhPTH (1-34)

Control

IRS-1 signal in PTH osteoanabolic

WT

action and bone fracture healing
Bone anabolic action of PTH has also

IRS-1–/–

been suggested to be mediated byinduction of IGF-I in osteoblasts, whichacts locally as an autocrine/ paracrine

IRS-2–/–

B

0.06
0.05

factor.21 To learn the involvement ofControlrhPTH (1-34)

mg/cm2*

IRS-1 and IRS-2 in the bone anabolic

0.03

action of PTH in vivo, IRS-1–/– and

WT IRS-1–/–
Femur
0.06
mg/cm2

IRS-2–/–mice and their respective

0.05

wild-type littermates were given daily

0.04
0.03

injections of PTH or vehicle for 4 weeks(Fig. 7).22 In the wild-type mice, thePTH injection increased bone mineraldensities of the femur, tibia, andvertebrae. These stimulations were

*

0.04

WT IRS-2–/–
Femur

*

WT

IRS-1–/–Tibia

WT IRS-1–/–
Vertebra

WT

IRS-2–/–Tibia

WT IRS-2–/–
Vertebra

Figure 7. Effects of in vivo injection of recombinanthuman (rh)PTH (1-34) in IRS-1–/– and IRS-2–/– mice.
The knockout mice and the respective wild-type (WT)
littermates underwent daily injections of rhPTH (80 g/kg)
or the vehicle for 4 weeks. (A) Representative toluidineblue stainings of proximal tibias. (B) Bone mineraldensities of the entire femurs, tibias, and vertebral bodies
determined by dual-energy X-ray absorptiometry. *P<0.01
vs. WT with rhPTH treatment.

9

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

similarly seen in IRS-2–/–mice; however, they were markedly suppressed in IRS-1–/–mice.
These results indicate that the PTH bone anabolic action is mediated by the activation of IRS-1,
but not IRS-2, as a downstream signal of IGF-I.22
A

0

B

1

2

3

6

10 wksTo further know the
involvement of IRS-1 in bone

WT

formation, we compared the
IRS-1–/–

healing of bone fracture
C

WT

which was created at themidshaft of mouse tibia andfixed with an intramedullary
nail in IRS-1–/– mice (Fig.

IRS-1–/–D

4

LY (Akt inhibitor)
PD (ERK inhibitor)
SB (p38 inhibitor)(X104 dpm)

3[3H]-TdR
uptake2

0*

WT mice showed bone union,*

while in IRS-1–/– mice, only*

1

Inhibitors (µM)
IGF-I (10 nM)

8A).23 After 3 weeks, all 15

0
(–)

0

1

3
(+)

WT

10

*
0
(–)

0

1

3

10

(+)
IRS-1–/–

Figure 8. (A) The mouse fracture model. (B) Time course of fracture
healing in representative WT and IRS-1–/– mice. (C) Radiographs offracture sites of all WT mice (n=15) and IRS-1–/– mice (n=15) 3 weeks
after the fracture. (D) Proliferation of primary osteoblasts determined by[3H]-TdR uptake by IGF-I and specific inhibitors. *p<0.01 vs. IGF-I
alone.

four showed bone union but
with small fracture callus,
and the other 11 miceremained non-union (Fig. 8B& C). Contrarily, the fracture healing was not suppressed in IRS-2 mice (data not shown). In theex vivo cultures of WT osteoblasts, cell proliferation stimulated by IGF-I was suppressedstrongly by an inhibitor of Akt signal, moderately by an ERK inhibitor, but not by an p38 MAPK
inhibitor (Fig. 8D). Meanwhile, IRS-1–/– osteoblasts were less respondent to IGF-I and were10

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

inhibited only by the ERK inhibitor. These indicate that the osteoanabolic action of IGF-I/IRS-1
is mainly mediated by the Akt pathway.

Akt1 signal in bone formation and resorption
Akt, a phosphoinositide-dependent serine-threonine protein kinase, is one of the key players inthe signal of potent bone anabolic factors. Our analyses of the knockout mice with Akt1, a major
Akt in osteoblasts and osteoclasts, revealed that the mice exhibited osteopenia with decreased
both bone formation and resorption (Fig. 9A-C).24 Figure 9D summarizes the mechanisms

24

ex vivo cell culture analyses.
Briefly, Akt1 suppresses theB

WT

Akt1–/–

transcription factor FoxO3anuclear entry and the proapoptotic

function by enhancing the Runx2
transcriptional activity, resulting

0

Ob.S/BSTUNEL(+)

15

2

10

2
0

*
(%)

*

5
0

Oc.S/BS

ES/BS

6

6

200

*

4

100

2

0

0

*

4
2

**

1
0

300

*

WT
Akt1–/–0

Akt1
Osteoblasts

mitochondria-dependent apoptosis

stimulates the differentiation and

0.5

BFR
4

D

susceptibility to

Bim transactivation, and1*

N.Oc/B.Pm(/ 100mm)

which were obtained from furtherof osteoblasts by inhibiting the

MAR
1.5

(mm/day)

maintain bone mass and turnoverC(%)

Akt1–/–(%)

WT(mm3/cm2/y)A(%)

underlying the Akt1 function to

Apoptosis via
FoxO3a / Bim

Osteoclasts
RANKL

Differentiation

Runx2
transcriptional
activityCellautonomous
differentiation& survival

Bone formation

Bone resorption

Bone mass & turnover

Figure 9. (A) 3D-CT of distal femurs of WT and Akt1–/– littermatesat 8 weeks of age. (B) Calcein double labelings (bars, 20 mm) and
TRAP staining (bars, 100 mm) of proximal tibias of the two
genotypes. (C) Bone histomorphometric analysis of the proximal
tibiae. The upper row shows bone formation parameters and the lower
row shows bone resorption parameters. *p<0.05, **p<0.01 vs. WT.
(D) Schematic diagram of the mechanisms underlying the Akt1
function to maintain bone mass and turnover (Details are shown in ref.
24).

11

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

in increased bone formation. Akt1 also induces receptor activator of nuclear factor-kB ligand(RANKL), a major determinant of osteoclastogenesis, in osteoblasts to support osteoclastdifferentiation, and shows cell-autonomous effects in osteoclasts to stimulate the differentiationand survival, resulting in increased bone resorption.
Taken together, the insulin-IGF-I / IRS-1-IRS-2 / Akt1 signal may play crucial roles forbone formation under various physiological and pathological conditions. It maintains bonevolume and turnover for prevention of osteoporosis, enhances OPLL progression, mediates thePTH bone anabolic action, and facilitates bone fracture healing. Further understanding of themolecular network related to this signal will provide a basis for rational therapeutic targets forthese disorders.

Runx2 regulating cartilage degradation in OA
Osteoarthritis (OA), a chronic degenerative joint disorder characterized by articular cartilage
degradation and osteophyte formation, is a major cause of disability in aged people. Despite
significant social demand for more information, the approved risk factors have to date been
limited to age, obesity, trauma history, occupation, and gender.25 Since these factors are closely
related to the accumulation of mechanical loading to joints, mechanical instability of the joints
may play a major role in the OA pathogenesis. In efforts to clarify the mechanisms whereby thejoint instability leads to OA development, we have established mechanical instability-induced
OA experimental models in mice that are reproducible and resemble the human OA, using a
microsurgical technique.26 Our examination of the time course of histology of the mouse joint
cartilage using the OA model revealed that type X collagen (COL10), a specific marker of12

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

hypertrophic chondrocytes,

OA
2

appeared in the superficial and

4

Sham
8w

8w

Safranin-O

middle zones above the tidemark
at 4 weeks, and MMP13

MMP13

expression appeared in thehypertrophic chondrocytes at 8

COL10

weeks (Fig. 10).27 These findings
Runx2

suggest that articular chondrocytes
undergo hypertrophic
differentiation in response to joint
instability, and the hypertrophic

Figure 10. Time course of expressions of MMP13, COL10, andRunx2 in the medial tibial cartilage of OA-induced and shamoperated knee joints in the experimental mouse model.
Localization of MMP13 and COL10 was detected byimmunohistochemistry, and Runx2 localization was detected by Xgal staining of heterozygous Runx2 deficient mice with LacZ
knock-in at the site of Runx2 deletion (Runx2+/lacz). Arrowheads
indicate the level of tidemark.

chondrocytes express MMP13 thatmay degrade the cartilage matrix.
Since a transcriptional activator Runx2 is known to induce both chondrocyte hypertrophy
and MMP13 expression,28,29 we then examined the involvement of Runx2 during OA
development.27 Runx2 expression was induced above the tidemark in the cartilage as early as 2
weeks, enhanced at 4 weeks, and decreased thereafter by the OA induction, which was notobserved in the sham-operated cartilage (Fig. 10).
For the functional analyses of Runx2, we used heterozygous Runx2-deficient mice
(Runx2+/–), since homozygous Runx2-deficient (Runx2–/–) mice died just after birth. The
Runx2+/– mice showed normal skeletal development and articular cartilage under physiologicalconditions (data not shown). When the OA progression was compared between WT and13

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

Runx2+/– joints, theA

cartilage degradation in

4 wks

WT

Runx2+/–B

WT

Runx2+/–

Runx2+/– was much milder
than that of the WT cartilage

8 wks

at 8 weeks and thereafter(Fig. 11A).27 The Runx2+/–
joint also showed decreased
osteophyte formation (Fig.
11B). These findings
demonstrate that Runx2
contributes to cartilage
degradation and the

12 wks3

WT
Runx2+/–

3

Cartilage 2
destruction
score
1

*
0

4

8

*
12 wks2
Osteophyte
formation
score 1

WT
Runx2+/–*

0

Figure 11. (A) Safranin-O staining and the cartilage destruction score of themedial tibial cartilage. Arrowheads indicate the level of tidemark. (B)Anteroposterior X-ray features, Safranin-O staining, and the osteophyte
formation score 12 weeks after surgery. Means (symbols or bars) ± SEM(error bars). *P<0.01 vs. WT.

subsequent osteophyte formation under the joint instability.
In addition to hypertrophic differentiation of chondrocytes, chondrocyte apoptosis is known
to be involved in OA development.30,31 When we created the experimental OA medial model inthe hetero-knockout mice of osteoprotegerin, joint destruction was enhanced as compared to thewild-type littermates.32 On the contrary, an intraarticular injection of recombinantosteoprotegerin suppressed joint destruction with a decrease of apoptotic chondrocytes. Since
tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a ligand of osteoprotegerin,
induces chondrocyte apoptosis, osteoprotegerin might inhibit the apoptosis induced by TRAIL.
Pro-inflammatry factors like prostaglandins (PGs), tumor necrosis factor-α (TNF-α), andinterleukin-1 (IL-1), and IL-6 have been suggested to be involved in the OA development.33

14

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

However, our previous report showed that levels of TNF-α,

WT

mPGES-1–/–

IL-1, and IL-6 in the synovial fluid from knee joints of OA
patients were much lower than those of rheumatoid arthritis
patients.34 In addition, although we created the experimental
OA model in the knockout mice of microsomal PGES-1(mPGES-1), a terminal enzyme for the PGE2 synthesis, thecartilage degradation and osteophyte formation werecomparable to the wild-type littemates (Fig. 12).35 We
therefore believe that inflammation may be associated with

Figure 12. Cartilage degradation in
the medial portion of tibial cartilageof 8-week-old wild-type (WT) andmPGES–/– mice in the experimental
OA model (Safranin O staining). The
inset boxes indicate the regions of thelower panels. Bars, 500 μm (upper) &50 μm (lower).

the OA process as a consequence, but might not have a central role in the cause of OA initiationor progression.
Taken together, chondrocyte hypertrophy and apoptosis, which constitute endochondral
ossification, play some roles in cartilage degradation during OA development under mechanicalstress. Our recent search by the COL10 promoter analyses has so far identified several factors
including cyclic GMP-dependent protein kinase II and S100 proteins as potent inducers of
endochondral ossification, which may possibly be crucial for the OA development.36-40

Carminerin regulating osteophyte formation in OA
Considering that osteophyte of OA is formed by chondrocyte calcification during endochondral
ossification, we have been attempting to isolate molecules that are involved in this process. Our
differential display analysis identified a novel molecule that was up-regulated by a highphosphate diet in association with calcification of auricular cartilage in the naturally occurring

15

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

A

High Phosphatemouse mutant ttw (tiptoe walking) with

Control

generally enhanced calcification (Fig. 13A).41
This molecule, termed carminerin, was also
called cystatin 10 due to the amino acid
B
ki

ey
dn

ne

lu

ng

ge
ti
le
la
r art ain usc tis tes ne rti
e
s n
a
bo c
l i v h e b r m te i

Carminerin

about 40% homology to cystatin C. Expression

GAPDH
α-Cmr

C

sequence containing a cystatin consensus, with

pCMV-Cmr

Dof carminerin was specific to cartilage (Fig.

pCMV13B), mainly localized in the cytoplasm ofhypertrophic chondrocytes (Fig. 13C). Mousechondrogenic ATDC5 cells overexpressing
WT

Cmr–/–

F
Body length (cm)Ecarminerin showed an increased calcification,

10
8suggesting that carminerin promotes

6

WT
Cmr+/–
Cmr–/–

4
2
0

0

2

4

6

8 10 wks

Figure 13. (A) Radiographs of ttw mice showing
enhanced calcification in the auricular cartilage(arrowheads) by a high phosphate diet. (B) Tissue
expression of carminerin by RT-PCR. (C)Immunohistochemistry of carminerin in the growthplate of an embryonic mouse (E18.5). Blue, yellow,
and red bars indicate proliferative, prehypertrophic,
and hypertrophic layers, respectively. (D) Alizarin red
staining of carminerin-overexpressing pCMV-Cmr
and the control pCMV/ATDC5 cells. (E) The
skeleton of WT and Cmr–/– littermate embryos(E17.5) stained with Alizarin red and Alcian blue. (F)Growth curves of WT, Cmr+/– and Cmr–/– mice.

chondrocyte calcification (Fig. 13D). To
further investigate the in vivo role ofcarminerin, we generated mice lacking
carminerin (Cmr–/–), and found that the micedeveloped and grew similarly to WT andCmr+/– littermates without abnormality ofmajor organs (Fig. 13F & F).42

Radiological analyses, however, revealed that Cmr–/– mice experienced decreases intrabecular bones mainly at the metaphysis, as compared to the WT littermates at 8 weeks of age(Fig. 14A). When the OA model was created in the WT and Cmr–/– knee joints, the joint

16

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disordersA

WT

Cmr–/–

WT

B

cartilage degradation was

Cmr–/–

vonKossa

similarly visible at the posterior

X-ppart of the tibias (Fig. 14B,
3DCT

D

15
10
5
0
X-p

Osteophyte
volume (mm3)Mankin score

Ctop), and Mankin’s grading

TB

WT
Cmr–/–

4*2

score showed comparable
values (Fig. 14C, left) in both0von-Kossa

genotypes. However,
osteophyte formation at the

3DCT

+/+

posterior tibias shown by
von-Kossa staining (Fig. 14B,
top) and 3D-CT analysis (Fig.

–/–

14B, bottom; Fig. 14C, right)
Figure 14. (A) Radiographs and 3D-CT of femurs of WT and Cmr–/–littermates under physiological conditions. (B) von Kossa staining and3D-CT of knee joints of the two genotypes in the experimental OA
model. (C) Quantification of the cartilage destruction and theosteophyte formation in the OA model by Mankin score and theosteophyte volume measured on 3D-CT images, respectively. *P<0.01
vs. WT. (D) Radiographs, toluidine blue (TB) and von Kossa stainings
3 weeks after fracture at the tibias.

was significantly decreased.
These findings indicate thatcarminerin contributes toosteophyte formation without

affecting cartilage degradation.42 We further examined the involvement of carminerin in bone
fracture healing at the midshaft of tibias. Cmr–/– mice showed a bone gap on the radiograph 3
weeks after the fracture, with substantial soft callus formation but impaired calcification (Fig.
14D). This model confirms that the carminerin deficiency impaired endochondral ossification by
suppressing chondrocyte calcification.

17

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

Figure 15 summarizes our

Mechanical stress
Runx2

hypothessis of the molecular
backgrounds of OA progression under

Chondrocyte hypertrophy & apoptosis
Centralmechanical stress in joints. As the

MMP13, etc.

–

mechanism involved in this stresscausing production of proteinases like
MMP13, we hereby propose the

Periphery

Vascularity

Cartilage
degradation

Carminerin+
Osteophyte
formation

Figure 15. A schema of possible molecular backgrounds of
OA under mechanical stress.

importance of chondrocyte hypertrophy
and apoptosis, essential steps for endochondral ossification that occurs physiologically in thegrowth plate cartilage.43 The proteinases produced by hypertrophic chondrocytes cause cartilage
degradation at the center of the joint and osteophyte formation at the periphery. The difference ofthe two sites may depend on the vascularity. At the periphery, vascularity is accessible fromsynovium or tendon, which causes endochondroal ossification to occur and make osteophytes,
just like at the growth plate cartilage. Carminerin may play a role in the chondrocyte calcification
at this stage. However, in the center, the vascularity is not accessible from the edge, so that itends up with cartilage degradation without being replaced by bone.

Conclusion
This paper has introduced our mouse genetics studies on the molecular backgrounds ofosteoporosis, OPLL, fracture healing, and OA. In the mechanism of age-related osteoporosis, wehave proposed involvement of PPARγ and the IRS-1-IRS-2/Akt1 signal. These signals may workwith putative molecules like vitamin D, estrogen, Runx2 and other osteoanabolic cytokines to18

Mouse genetics studies on molecular mechanisms underlying bone and cartilage disorders

maintain bone mass. One of the next tasks ahead of us will be to elucidate the network system ofthese many factors. Bone formation seen in OPLL, PTH osteoanabolic action, and fracture
healing may also be mediated by the insulin-IGF-I / IRS-1 / Akt signal. For OA, Runx2 andcarminerin were shown to be involved in distinct OA features: cartilage degradation andosteophyte formation, respectively.
The ultimate aim of the present study is to identify the molecular targets for clinicaltreatments of the degenerative skeletal disorders. Although we mainly used the mouse genetics
approaches, we have attempted to confirm the reproducibility of the mouse findings in humans
using human gene polymorphism or clinical biochemical studies.44-49 Among the molecules we
identified in this study, there are some whose suppression ameliorated skeletal disorders underpathological conditons but did not affect physiological conditions, indicating that targeting onthese molecules may lead to an ideal treatment without side effects on physiological functions. In
fact, trials based on the present findings are being practically planned for clinical application.

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